Without limiting the scope of the invention, its background is described in connection with generation of cardiac cell lines and tissues for research, drug screening and autologous cell-based therapy by in vitro culturing of differentiable cells and other related methods.
One such method is taught in PCT Patent Application No. PCT/US2011/056329, filed by Singla, et al., entitled, Cardiac Induced Pluripotent Stem Cells And Methods Of Use In Repair And Regeneration Of Myocardium. Briefly, this application is said to disclose iPS cells from cardiac or ventricular specific cell types that are generated having the potential to repair and regenerate infarcted myocardium. The cells were transduced with four sternness factors and reprogrammed them into iPS cells. These cardiac iPS cells were able to differentiate into beating cardiac myocytes, formed cardiac-specific structures, and positively stained for cardiac specific proteins. Transplanted cells also significantly inhibited apoptosis and fibrosis and improved cardiac function.
Another application is U.S. Patent Application No. 2011/097799, filed by Stankewicz, et al., entitled, Cardiomyocyte Production. Briefly, methods and composition for the production of cardiomyocytes from differentiation of pluripotent stem cells are disclosed. For example, in certain aspects methods including differentiating pluripotent stem cells in a large volume of suspension culture in the presence of Rho-associated (ROCK) inhibitors are described. Also, methods for differentiation of stem cells into cardiomyocytes that overcome variability between different stem cell clones and different batch of culture medium are said to be provided.
Another such technique for generating cells predominantly displaying at least one characteristic associated with a cardiac phenotype is disclosed in U.S. Patent Application Publication No. 2005/037489 (Gepstein et al. 2005) (hereinafter Gepstein). The Gepstein invention comprises the steps of (a) partially dispersing a confluent cultured population of human stem cells, thereby generating a cell population including cell aggregates; (b) subjecting said cell aggregates to culturing conditions suitable for generating embryoid bodies; (c) subjecting said embryoid bodies to culturing conditions suitable for inducing cardiac lineage differentiation in at least a portion of the cells of said embryoid bodies, said culturing conditions suitable for inducing cardiac lineage differentiation including adherence of said embryoid bodies to a surface, and culture, medium supplemented with serum, thereby generating cells predominantly displaying at least one characteristic associated with a cardiac phenotype.
U.S. Patent Application Publication No. 2005/0054092 (Xu and Gold, 2005) provides a method of obtaining populations of human cells of the cardiomyocyte lineage. The cells are said to be obtained by causing cultures of pluripotent stem cells to differentiate in vitro, and then harvesting cells with certain phenotypic features. Differentiated cells bear cell surface and morphologic markers characteristic of cardiomyocytes, and a proportion of them undergo spontaneous periodic contraction. Highly enriched populations of cardiomyocytes and their replicating precursors can be obtained, and are suitable for use in a variety of applications, such as drug screening and therapy for cardiac disease.
A procedure for generating cardiomyocyte lineage cells from embryonic stem cells for use in regenerative medicine is described in U.S. Pat. No. 7,452,718 issued to (Gold et al. 2008) (hereinafter the '718 patent). The '718 patent describes a method that does not require differentiating by way of embryoid body formation or in serum. Instead, the stem cells are plated onto a solid substrate, and differentiated in the presence of select factors and morphogens. After enrichment for cells with the appropriate phenotype, the cells are allowed to cluster into cardiac Bodies™, which are remarkably homogeneous and suitable for the treatment of heart disease.
U.S. Patent Application Publication No. 2009/0017465 (Xu, 2009) provides populations human cells of the cardiomyocyte lineage. The cells are obtained by causing cultures of pluripotent stem cells to differentiate in vitro, and then harvesting cells with certain phenotypic features. Differentiated cells bear cell surface and morphologic markers characteristic of cardiomyocytes, and a proportion of them undergo spontaneous periodic contraction. Highly enriched populations of cardiomyocytes and their replicating precursors can be obtained, suitable for use in a variety of applications, such as drug screening and therapy for cardiac disease.
Finally, U.S. Pat. No. 7,611,852 issued to (Thomson et al., 2009) discloses human embryonic stem cells form embryoid bodies in culture, which contain differentiated human cells. Some of the human cells in embryoid bodies differentiate into cardiomyocytes. Here the biological and electrical characteristics of those cardiomyocytes are described with reference to the use of cardiomyocytes derived from human embryonic stem cells in drug screening protocols for mechanisms of cardiac toxicity.